Anti-vegfa fusion construct as well as preparation method therefor and use thereof

Abstract

Provided is an anti-VEGFA fusion construct. The fusion construct is formed by linking an anti-VEGFA antibody or an antigen-binding fragment thereof with an anti-albumin antibody or an antigen-binding fragment thereof. The fusion construct has strong VEGFA binding affinity and biological activity for inhibiting HUVEC proliferation stimulated by VEGFA, and the functions of domains do not affect each other. Also provided are a method for preparing the fusion construct, and the use thereof in the preparation of a product for treating and / or preventing diseases. The anti-VEGFA antibody or the antigen-binding fragment thereof has the activity of treating and / or diagnosing diseases.

Description

An anti-VEGFA fusion construct and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-VEGFA fusion construct and a preparation method and application thereof. Background Art

[0002] Vascular endothelial growth factor A (VEGFA), a key member of the VEGF family, is a major angiogenic factor that promotes endothelial cell differentiation, proliferation, migration, and invasion, and regulates blood vessel formation and development. VEGFA promotes angiogenesis primarily by binding to and activating the receptor VEGFR2 (VEGF Receptor 2) and its downstream pathways. Binding of VEGFA to the extracellular domain of VEGFR2 leads to receptor dimerization, protein kinase activation, tyrosine phosphorylation, and activation of downstream signaling pathways, stimulating endothelial cell proliferation and increasing vascular permeability. Activation of the VEGF signaling pathway occurs in a variety of diseases, ranging from cancer and autoimmunity to retinal diseases. Inhibiting the VEGF signaling pathway can treat diseases associated with abnormal vascular proliferation, such as wet age-related macular degeneration (wAMD). Currently, several anti-VEGF protein drugs are marketed for the treatment of tumors and diseases related to ocular neovascularization.

[0003] The first anti-VEGFA protein drug is the monoclonal antibody bevacizumab (Avastin). Other anti-VEGFA protein drugs include ranibizumab (Lucentis), aflibercept, and brolucizumab (Beovu). Among them, the half-life of free aflibercept in human aqueous humor is approximately 11 days, which is longer than ranibizumab (7 days) and bevacizumab (9 days). The dosing interval of aflibercept is once every 2 months. Currently, the dose of aflibercept has been increased from 2 mg to 8 mg, and clinical trials have shown that the dosing interval can be further extended to once every 3 or 4 months. Brolucizumab (Beovu) can be prepared as a 120 mg / mL protein solution, and the amount of protein injected into the vitreous cavity can reach 6 mg, which is the maximum dose currently available for a single injection. In the brolucizumab dosing regimen, patients received an injection once a month for the first three months, and then once every three months. The visual acuity improvement effect was no worse than that of the control group, aflibercept (injected once every two months). These anti-VEGF protein drugs are administered by intravitreal injection, with a volume of no more than 100 μL. The longest dosing interval for brolucizumab is three months, and the dosing interval for the other drugs is once every 1 to 2 months. Frequent intraocular injections increase the risk of increased intraocular pressure, intraocular inflammation, retinal detachment, etc., so there is an urgent need for long-acting anti-VEGFA therapeutic drugs in clinical practice to reduce the number of injections for patients.

[0004] Existing methods for reducing dosing frequency include increasing the concentration of protein drugs, thereby increasing the dose of a single injection; other methods include enhancing the biological activity of anti-VEGFA antibodies, lowering the minimum effective concentration, or increasing the half-life of the drug. While the development of long-acting anti-VEGFA therapeutics has achieved some clinical success, there are also some challenges. For example, high-concentration protein preparations often increase viscosity, susceptibility to protein aggregate formation, or technical issues such as poor stability, which increase the risk of intravitreal injections; Furthermore, there are issues such as insufficient biological effects or insignificant half-life enhancement. Therefore, the design and development of anti-VEGFA drugs requires further research and improvement.

[0005] Heavy chain antibodies are IgG2 and IgG3 antibodies that originate from the serum of camelids (camels, dromedaries, and llamas, etc.) and contain only heavy chains but no light chains. The variable domain of heavy chain (VHH) of heavy chain antibodies has independent antigen-binding activity and is the smallest functional fragment of natural antibodies. It is called a nanobody or single-domain antibody. Nanobodies have a molecular weight of approximately 13kDa, high thermal stability and good water solubility, and have great potential for development into higher-concentration protein preparations. Albumin is the most abundant protein in plasma (at a concentration of approximately 40mg / mL). It is crucial for maintaining plasma osmotic pressure. At the same time, albumin is an important transport carrier for endogenous ligands (such as fatty acids, metal ions, hormones, etc.) and exogenous ligands (such as drugs).

[0006] Albumin has a molecular weight of 66.5kDa and can bind to the cell surface receptor FcRn (neonatal Fc receptor) under weakly acidic conditions, but has a weaker binding force under neutral conditions. Therefore, after albumin binds to FcRn, it is internalized into the endosome. As the endosomal environment becomes acidic, the binding force between albumin and FcRn increases, and it is brought back to the cell surface by FcRn, or transported across cells. Therefore, albumin can achieve a longer half-life with the help of the recycling mechanism of FcRn. The half-life of human albumin in plasma is about 3 weeks. Therefore, small molecule compounds that bind to albumin, or proteins expressed by fusion with albumin (such as marketed drugs) and ), has significantly improved pharmacokinetic properties, obtains a longer half-life, can potentially extend the dosing interval, and provide patients with a better medication regimen.

[0007] Anti-albumin antibodies or antigen-binding fragments thereof can bind to albumins of different species with high affinity. At the same time, the anti-albumin antibodies can be linked to bioactive effector molecules, such as anti-VEGFA antibodies or antigen-binding fragments, to form fusion constructs. Fusion constructs can improve pharmacokinetic properties by binding to albumin, resulting in a longer half-life. However, the design of the fusion construct's structure, the connection between the various domains, and whether binding to albumin will affect the activity of the bioactive effector molecule still require a great deal of creative work. This application hopes to utilize nanobody technology for the development of anti-albumin antibodies or antigen-binding fragments, as well as the development and design of anti-VEGFA fusion constructs, to provide a superior anti-VEGFA fusion construct. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present application uses nanobodies that bind to VEGFA as the basic active unit, and designs monovalent and bivalent nanobodies to prepare anti-VEGFA fusion proteins with good stability and strong biological activity. In addition, the anti-VEGFA antibody or antigen-binding fragment is connected to an antibody or antigen-binding fragment of another antigen, such as an anti-albumin antibody, through a linker to prepare a fusion construct of the anti-VEGFA antibody or antigen-binding fragment and the anti-albumin antibody or antigen-binding fragment. This fusion construct has strong VEGFA binding affinity and biological activity that inhibits VEGFA-stimulated HUVEC proliferation. In addition, the functions of the various domains in the fusion construct do not affect each other. Specifically,

[0009] In a first aspect of the present invention, an anti-VEGFA fusion construct is provided, wherein the fusion construct comprises an anti-albumin antibody or an antigen-binding fragment thereof and an anti-VEGFA antibody or an antigen-binding fragment thereof, wherein the anti-VEGFA antibody or an antigen-binding fragment thereof is linked to the anti-albumin antibody or an antigen-binding fragment thereof.

[0010] Preferably, the anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region.

[0011] The amino acid sequence of CDR-H1 comprises SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1;

[0012] the amino acid sequence of CDR-H2 comprises SEQ ID NO:41 (GISVX1X2SFLDYADAVKG) or an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:41;

[0013] The amino acid sequence of CDR-H3 comprises SEQ ID NO: 3 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 3.

[0014] Wherein, X in SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG) can be any natural amino acid residue, such as alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), and valine (V).

[0015] In a specific embodiment of the present invention, X1X2 in SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG) represents DS, DA, EG or DG.

[0016] In a specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups (see Table 42 for details):

[0017] A) SEQ ID NO: 1, 2, 3;

[0018] B) SEQ ID NO: 1, 10, 3;

[0019] C) SEQ ID NO: 1, 11, 3;

[0020] D) SEQ ID NO: 1, 12, 3.

[0021] Table 42 Anti-albumin candidate antibody CDR sequences

[0022] Preferably, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from N-terminus to C-terminus.

[0023] The anti-albumin antibody or antigen-binding fragment thereof comprises a heavy chain variable region. In this application, the division of the antibody CDR region amino acids adopts the Kabat numbering system.

[0024] Preferably, the anti-albumin antibody or antigen-binding fragment thereof comprises a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region. Further preferably, the modification site of the humanized sequence is located in the framework region of the variable region and / or the constant region.

[0025] Preferably, the structure of the anti-albumin antibody or its antigen-binding fragment includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single chain antibody (scFv) or a linear antibody.

[0026] The anti-albumin antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.

[0027] In a specific embodiment of the present invention, the anti-albumin antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibodies, Fab, and scFv, nanobodies have a higher molar concentration at the same mass and can bind to more antigen molecules.

[0028] The anti-albumin antibody or antigen-binding fragment thereof can bind to mammalian albumin.

[0029] Preferably, the mammals include humans or non-human mammals, and the non-human mammals may be wild animals, zoo animals, commercial animals, pets, experimental animals, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0030] Preferably, the amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 4, 6-9, 13-15, or has at least 80% identity with any one of SEQ ID NOs: 4, 6-9, 13-15.

[0031] In a specific embodiment of the present invention, the amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID NOs: 4, 6-9, 13-15.

[0032] The anti-albumin antibody or antigen-binding fragment thereof can be constructed using any conventional method in the prior art, such as artificial synthesis or eukaryotic or prokaryotic expression.

[0033] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region;

[0034] wherein the amino acid sequence of CDR-H1 comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16;

[0035] the amino acid sequence of CDR-H2 comprises SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 17;

[0036] The amino acid sequence of CDR-H3 comprises SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 18.

[0037] The anti-VEGFA antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0038] The structure of the anti-VEGFA antibody or its antigen-binding fragment includes nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fv fragment, dAb fragment, F(ab')2 fragment, single chain antibody (scFv) or linear antibody.

[0039] The anti-VEGFA antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.

[0040] The anti-VEGFA antibody or antigen-binding fragment thereof can be a humanized antibody or a fully human antibody.

[0041] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region. Further preferably, the modification site of the humanized sequence is located in the framework region of the variable region and / or the constant region.

[0042] In a specific embodiment of the present invention, the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibodies, Fab, and scFv, nanobodies have a higher molar concentration at the same mass and can bind to more antigen molecules.

[0043] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof can bind to human or monkey VEGFA protein.

[0044] Preferably, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 19-28, or has at least 80% identity with any one of SEQ ID NOs: 19-28.

[0045] Preferably, the fusion construct comprises one or more anti-VEGFA antibodies or antigen-binding fragments thereof.

[0046] Preferably, the fusion construct comprises one or more anti-albumin antibodies or antigen-binding fragments thereof.

[0047] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof is directly or indirectly linked to the anti-VEGFA antibody or antigen-binding fragment thereof.

[0048] Preferably, the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are directly or indirectly linked.

[0049] Preferably, the anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly connected to the anti-albumin antibody or antigen-binding fragment thereof.

[0050] Preferably, the indirect connection is through a linker, a functional domain and / or a linker for coupling.

[0051] Wherein, the linker is selected from connecting peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty chains, biotin, streptavidin or avidin.

[0052] The functional domain is a combination of one or more of an Fc fragment, serum albumin, cytokine, transferrin or scaffold protein.

[0053] The linker used for coupling includes a functional group linker. Preferably, the functional group linker includes a thiol, amino, hydroxyl and / or carboxyl reactive group, which can covalently couple the anti-albumin antibody or its antigen-binding fragment with the anti-VEGFA antibody or its antigen-binding fragment.

[0054] Preferably, the N-terminus and / or C-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof is connected to the C-terminus and / or N-terminus of another anti-VEGFA antibody or antigen-binding fragment thereof via a connecting peptide.

[0055] In a specific embodiment of the present invention, the sequence from N-terminus to C-terminus includes a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, and a second anti-VEGFA antibody or an antigen-binding fragment thereof.

[0056] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof is directly or indirectly linked to the N-terminus, C-terminus and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.

[0057] In a specific embodiment of the present invention, the fusion construct comprises two anti-VEGFA antibodies or antigen-binding fragments thereof, and the two anti-VEGFA antibodies or antigen-binding fragments thereof are directly or indirectly linked to the N-terminus, C-terminus and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.

[0058] For example, in the fusion construct, the order of connection of the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof from N-terminus to C-terminus is as follows:

[0059] 1) anti-albumin antibody or antigen-binding fragment thereof, connecting peptide, anti-VEGFA antibody or antigen-binding fragment thereof;

[0060] 2) anti-VEGFA antibodies or antigen-binding fragments thereof, connecting peptides, anti-albumin antibodies or antigen-binding fragments thereof;

[0061] 3) an anti-albumin antibody or an antigen-binding fragment thereof, a connecting peptide, a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, a second anti-VEGFA antibody or an antigen-binding fragment thereof; or,

[0062] 4) a first anti-VEGFA antibody or antigen-binding fragment thereof, a connecting peptide, an anti-albumin antibody or antigen-binding fragment thereof, a connecting peptide, a second anti-VEGFA antibody or antigen-binding fragment thereof, and the like;

[0063] Wherein, the connection may not include a connecting peptide.

[0064] Preferably, the two or more anti-VEGFA antibodies or antigen-binding fragments thereof are anti-VEGFA antibodies or antigen-binding fragments thereof with completely identical sequences, anti-VEGFA antibodies or antigen-binding fragments thereof with partially identical sequences, or anti-VEGFA antibodies or antigen-binding fragments thereof with completely different sequences.

[0065] In a specific embodiment, the fusion construct comprises any one of the amino acid sequences in SEQ ID NOs: 29-35, or has at least 80% identity to any one of the amino acid sequences in SEQ ID NOs: 29-35.

[0066] Preferably, the fusion construct comprises an Fc fragment.

[0067] Further preferably, the Fc fragment comprises SEQ ID NO: 38, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 38.

[0068] Preferably, the fusion construct further comprises a secretory peptide. The secretory peptide is connected to the N-terminus of the fusion construct. Further preferably, the amino acid sequence of the secretory peptide may be SEQ ID NO: 36.

[0069] Preferably, the fusion construct further comprises a tag.

[0070] Preferably, the tag is connected to the C-terminus of any antibody or antigen-binding fragment thereof, or fusion construct.

[0071] Preferably, the fusion construct can inhibit or compete with the binding of other anti-VEGFA antibodies (preferably antibodies that bind to the same or overlapping epitopes as the anti-VEGFA antibodies of the present invention) to VEGFA.

[0072] Preferably, the fusion construct can also inhibit or compete for the binding of other anti-albumin antibodies (preferably antibodies that bind to the same or overlapping epitopes as the anti-albumin antibodies of the present invention) to albumin.

[0073] A second aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment described herein, or the fusion construct described above. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid is a therapeutic nucleic acid.

[0074] In some embodiments, the nucleic acid is DNA, which encodes the antibody or antigen-binding fragment described herein, or the fusion construct described above.

[0075] Preferably, the nucleotide sequence encoding CDR-H1 in the anti-albumin antibody or antigen-binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 42 or 51 or a degenerate sequence thereof, or has at least 80% identity with any one of the nucleotide sequences in SEQ ID NO: 42 or 51.

[0076] Preferably, the nucleotide sequence encoding CDR-H2 in the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 43, 52-54 or a degenerate sequence thereof, or has at least 80% identity with any one of SEQ ID NOs: 43, 52-54.

[0077] Preferably, the nucleotide sequence encoding CDR-H3 in the anti-albumin antibody or antigen-binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 44 or 55 or a degenerate sequence thereof, or has at least 80% identity with any one of the nucleotide sequences in SEQ ID NO: 44 or 55.

[0078] Preferably, the nucleotide sequence encoding CDR-H1 in the anti-VEGFA antibody or antigen-binding fragment thereof comprises any nucleotide sequence in SEQ ID NO: 59 or a degenerate sequence thereof, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 59.

[0079] Preferably, the nucleotide sequence encoding CDR-H2 in the anti-VEGFA antibody or antigen-binding fragment thereof comprises any nucleotide sequence in SEQ ID NO: 60 or a degenerate sequence thereof, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 60.

[0080] Preferably, the nucleotide sequence encoding CDR-H3 of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any nucleotide sequence in SEQ ID NO: 61 or a degenerate sequence thereof, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 61.

[0081] More preferably, the nucleotide sequence encoding the anti-VEGFA antibody or its antigen-binding fragment comprises any one of SEQ ID NOs: 62-71 or a degenerate sequence thereof, or a nucleotide sequence that has at least 80% identity with any one of SEQ ID NOs: 62-71 and has the function of encoding an anti-VEGFA antibody or its antigen-binding fragment.

[0082] More preferably, the nucleotide sequence encoding the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 45, 47-50, 56-58 or a degenerate sequence thereof, or a nucleotide sequence that has at least 80% identity with any one of SEQ ID NOs: 45, 47-50, 56-58 and has the function of encoding an anti-albumin antibody or an antigen-binding fragment thereof.

[0083] More preferably, the nucleotide sequence encoding the above-mentioned fusion construct comprises any one of the nucleotide sequences in SEQ ID NOs: 72-78 or a degenerate sequence thereof, or has at least 80% identity with any one of the nucleotide sequences in SEQ ID NOs: 72-78, and has a nucleotide sequence encoding the function of the above-mentioned fusion construct.

[0084] In some embodiments, the nucleic acid is mRNA. One or more modification techniques can be used to produce more stable mRNA. Known mRNA modification technologies can be roughly divided into three categories: using artificially synthesized non-natural ribonucleic acids instead of natural ribonucleic acids to synthesize mRNA; adding 5'caps, 3'poly (A) "tails" and UTR (untranslated region) sequences; using special new formulation technologies to effectively protect mRNA. Among them, the preferred mRNA modification technology can synthesize mRNA by replacing natural ribonucleic acids with artificially synthesized non-natural ribonucleic acids. Chemical modifications on eukaryotic mRNA can be roughly divided into three categories: methylation, pseudouridine (Ψ) and hypoxanthine. For example, the chemical modification can be selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0085] The third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

[0086] The vector can be expressed in vivo, in vitro or ex vivo. Preferably, the vector is a recombinant vector such as a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector.

[0087] For example, a viral expression vector can be used. The viral expression vector can include viral-derived DNA or RNA sequences for packaging into viruses (such as retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV). The virus and viral expression vector can be used for in vitro, ex vivo, and / or in vivo delivery. For example, the recombinant vector can be included in a delivery vector. In certain embodiments, the delivery vector includes antibody or Fab as described herein, nucleic acid as described herein, and / or recombinant vector as described herein, and optionally includes liposomes and / or lipid nanoparticles (LNPs). For example, delivery vectors can be introduced into cells by physical delivery methods. The example of physical method includes microinjection, electroporation, and hydrodynamic delivery. For example, nucleic acid as described herein can be wrapped in cationic lipid particles (such as liposomes) by LNPs, and can be delivered to cells relatively easily.

[0088] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid or the above-mentioned vector.

[0089] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0090] Eukaryotic cells include animal and plant cells, fungi, etc., such as T cells, yeast cells, HEK293 cells or CHO cells, etc.

[0091] Prokaryotic cells such as Escherichia coli.

[0092] The fifth aspect of the present invention provides a method for preparing a host cell comprising the above-mentioned nucleic acid or the above-mentioned vector, wherein the preparation method comprises introducing the above-mentioned nucleic acid or vector into the host cell.

[0093] In a sixth aspect, the present invention provides a method for preparing the above-mentioned fusion construct, which comprises culturing the above-mentioned host cell to express the fusion construct.

[0094] In a seventh aspect, the present invention provides a product for treating and / or diagnosing a disease, wherein the product for treating and / or diagnosing a disease comprises any one of the following:

[0095] A) The fusion construct described above;

[0096] B) the nucleic acid described above;

[0097] C) the above-mentioned vector; or,

[0098] D) The host cell described above.

[0099] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof has activity in treating and / or diagnosing diseases.

[0100] Preferably, the product can be a diagnostic kit, a drug, or a diagnostic chip, etc.

[0101] The diseases are diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0102] Preferably, the fusion construct blocks VEGFA-mediated vascular endothelial cell proliferation or inhibits angiogenesis.

[0103] In an eighth aspect, the present invention provides a method for detecting VEGFA, comprising contacting a sample to be tested with the above-mentioned fusion construct, and then detecting the content of a complex formed between VEGFA and the above-mentioned fusion construct.

[0104] The detection method is to detect the presence or content of VEGFA, wherein the presence refers to the presence or absence, and the content can be the expression level or protein concentration.

[0105] Preferably, the VEGFA is from a mammal, more preferably, the mammal is from a human, mouse or monkey.

[0106] The ninth aspect of the present invention provides a method for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, the method comprising contacting vascular endothelial cells with the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing a disease.

[0107] Preferably, the method comprises diluting the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing a disease with a serum-free culture medium of vascular endothelial cells, and then incubating with an antigen (eg, VEGFA165).

[0108] Preferably, after incubation, the method comprises the step of discarding the complete culture medium of the vascular endothelial cells in the culture plate.

[0109] Preferably, the antigen and the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing a disease are mixed with vascular endothelial cells, added to a vascular endothelial cell culture plate, and cultured on the culture plate.

[0110] Preferably, the detection is performed after culturing.

[0111] Preferably, the incubation time is 0.5-5 h, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h.

[0112] Preferably, the incubation temperature is room temperature-45°C, preferably 30-40°C, for example 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc.

[0113] Preferably, the culture temperature is room temperature-45° C., preferably 30-40° C., such as 25, 30, 35, 36, 37, 38, 39, 40, 45° C., etc. Preferably, the culture is carried out in an incubator with 5% CO 2 .

[0114] Preferably, the culture time is 24-120 hours, preferably 48-96 hours, for example 24, 36, 48, 68, 72, 96, 120 hours, etc.

[0115] Preferably, the detection is the detection of the number of living vascular endothelial cells.

[0116] The tenth aspect of the present invention provides a method for treating and / or preventing a disease, which comprises administering the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, and the above-mentioned product for treating and / or diagnosing the disease to an individual.

[0117] The diseases include diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0118] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0119] In the eleventh aspect of the present invention, the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector or the above-mentioned host cell is used in the preparation of a product for treating and / or preventing a disease, and the anti-VEGFA antibody or its antigen-binding fragment has the activity of treating and / or diagnosing a disease.

[0120] The diseases include diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0121] Preferably, the product includes a product that blocks VEGFA-mediated vascular endothelial cell proliferation or inhibits angiogenesis.

[0122] The product described in this application may be a kit, a drug, a chip, an antibody-drug conjugate, etc.

[0123] As used herein, a "fusion construct" defines a fusion of an anti-albumin antibody or antigen-binding fragment thereof of the present invention with another compound. The fusion construct may comprise one or more anti-albumin antibodies or antigen-binding fragments thereof, and the multiple anti-albumin antibodies or antigen-binding fragments thereof may be the same or different. The fusion construct may comprise one or more additional compounds, and the multiple additional compounds may be the same or different. The compounds may be proteinaceous compounds or non-proteinaceous compounds. Where the compounds are proteinaceous compounds or the fusion construct comprises only multiple anti-albumin antibodies or antigen-binding fragments thereof, the fusion construct may also be referred to as a fusion protein. Where the compound is fused to the anti-albumin antibody or antigen-binding fragment thereof in a conjugated form, the fusion construct may also be referred to as a conjugate.

[0124] The "medicine" of the present invention can be used to treat humans or non-human animals, such as non-human mammals. The medicine can include pharmaceutically acceptable carriers, excipients or salts common in the prior art. The medicine can be administered by any suitable route, such as enteral administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.). The medicine can be in any suitable dosage form, such as enteral administration or parenteral administration, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc. The various dosage forms of the medicine can be prepared according to the conventional production methods of pharmaceutical field.

[0125] The drug may contain 0.01-99.5% (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the fusion construct, the nucleic acid, the vector, the host cell, and the like by weight. The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL).

[0126] The single dose of the drug can be 0.1-1000 mg, for example, 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.

[0127] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0128] The "method" described in the present invention can be used for the diagnosis and treatment of diseases or for non-disease diagnosis and treatment purposes.

[0129] The "antigen-binding fragment" of the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody that can specifically bind to the epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or a single or two or more CDRs from the heavy chain or light chain of an antibody. Among them,

[0130] Nanobodies or single-domain antibodies refer to the variable domain of heavy chain (VHH) of an antibody, which has independent antigen-binding activity.

[0131] Chimeric antibodies are antibodies in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0132] Single-chain antibodies are antibodies composed of the variable regions of the heavy chain and the light chain connected by a linker peptide.

[0133] Fab, a monovalent fragment consisting of the VL, VH, CL and CH1 domains.

[0134] Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0135] F(ab')2 is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0136] Fd, Fd fragment consisting of VH and CH1 domains.

[0137] Fv, an Fv fragment consisting of the VL and VH domains of a single antibody arm.

[0138] dAb fragments are antibody fragments composed of the VH domain.

[0139] The “linear antibody” described in the present invention comprises one or more pairs of antibody fragments connected in series, wherein the antibody fragment can be an Fd segment (VH-CH1), a single-chain antibody (scFv), an antibody fragment (Fab), or a single-domain antibody (VHH), and these fragments are connected in series through connecting peptides to form a continuous antibody structure.

[0140] Among them, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain constant region, and CH represents the heavy chain constant region.

[0141] The "Fc" region of the present invention contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments form a dimer by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.

[0142] The terms "comprising" or "including" as used in the present invention are open-ended. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0143] The "homology" or "identity" mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 112%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology / identity.

[0144] The "humanized antibody" of the present invention refers to an antibody whose framework region and / or constant region portion (e.g., CH region) or all or part of an antibody is encoded by a human antibody gene. In one embodiment of the present invention, the CDR region of the antibody has not been humanized.

[0145] The "individual" described in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, a commercial animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0146] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0147] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0148] The term "diagnosis" as used herein refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression or possible future progression of a disease.

[0149] As used herein, a "tumor" can be any undesirable cell proliferation (or any disease manifesting as undesirable cell proliferation), neoplasm, or a predisposition or increased risk of undesirable cell proliferation, neoplasm, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, leukocytes. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, neurilemoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0150] The term "retinal vascular disease" as used herein refers to a general term for diseases that occur in the retinal arteries or veins, or diseases associated with choroidal neovascularization. These diseases include, but are not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, and neovascular glaucoma.

[0151] In summary, the beneficial technical effects of the present invention include:

[0152] 1) The anti-VEGFA fusion construct obtained by the present invention has a strong VEGFA binding affinity (SPR test, K D=2.4-5.4pM), which is much higher than existing VEGFA antibody drugs and can compete with VEGFR2 for binding to VEGFA.

[0153] 2) The fusion construct inhibited VEGFA-stimulated HUVEC proliferation with an IC50 value similar to that of the receptor-based drug aflibercept. In VEGFA-stimulated reporter cells, the fusion construct inhibited luciferase expression and luciferin luminescence. In HUVEC cells, the fusion construct also inhibited VEGFA-activated VEGFR2 phosphorylation.

[0154] 3) The functions of the various domains in the fusion construct do not affect each other. The fusion construct of the present invention connects the anti-albumin antibody and the anti-VEGFA antibody and still has a strong binding affinity to human albumin (SPR test, K D =35-69 nM) and VEGFA binding affinity (tested by SPR, K D =2.4-5.4pM).

[0155] 4) In vivo, the fusion construct has the potential to inhibit VEGFA-stimulated angiogenesis. By binding to albumin, it is carried by albumin into specific albumin-rich tissue microenvironments, potentially enhancing the selectivity of the fusion construct for delivery to specific tissues. Furthermore, since the fusion construct increases in molecular weight upon albumin binding, it can participate in the binding and recycling mechanisms of albumin to FcRn, thereby extending its half-life. Therefore, the fusion construct of an anti-VEGFA antibody and an anti-albumin antibody has the potential to achieve long-lasting effects in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0157] Figure 1: Cootisol-stained gel images of His-tagged antibodies Alb Nb and 3005 after Ni column purification. (A) is a Cootisol-stained gel image of antibody Alb Nb, and (B) is a Cootisol-stained gel image of antibody 3005. The supernatant of the cell culture medium after transient transfection of antibody Alb Nb is labeled as Input, the flow-through that cannot bind to the Ni column is labeled as FT, the antibody Alb Nb that binds to the Ni column and is eluted is labeled as E, and the marker is labeled as M.

[0158] Figure 2: ELISA test of antibody 3005-Fc and Alb Nb-Fc binding to albumins from different species, (A) is HSA binding test, (B) is MSA binding test, and (C) is rat albumin binding test.

[0159] Figure 3: A stained gel image of the His-tagged 3005 humanized antibody after Ni column purification.

[0160] Figure 4: ELISA binding activity test of antibody 3005 and its humanized antibody binding to MSA.

[0161] Figure 5: ELISA binding activity test of antibody 3005, its humanized antibody 3005Hz6 and control antibody Alb Nb binding to HSA and rat albumin, (A) is HSA binding test, (B) is rat albumin binding test.

[0162] Figure 6: Binding and dissociation curves of humanized antibody 3005Hz6 with HSA and MSA (SPR method), (A) is the binding and dissociation curves of antibody 3005Hz6 with HSA, (B) is the binding and dissociation curves of antibody 3005Hz6 with MSA.

[0163] Figure 7: SDS-PAGE gel images of the purified humanized antibody 3005Hz6 mutant proteins 3005Hz6(EG), 3005Hz6(DS) and 3005Hz6(DA). (A) is an SDS-PAGE gel image of 3005Hz6(EG) and 3005Hz6(DS). The supernatant of the culture medium of cells transiently expressing 3005Hz6(EG) and 3005Hz6(DS) is marked as Input, the flow-through that cannot bind to the Ni column is marked as FT, and the eluted purified product is marked as E. (B) is an SDS-PAGE gel image of 3005Hz6(DA).

[0164] Figure 8: ELISA activity detection of humanized antibody 3005Hz6 and its mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) binding to HSA.

[0165] Figure 9: SDS-PAGE gel images of the fusion constructs V1SA-3005Hz6, 3005Hz6-V1SA and V1DP-3005Hz6 after Ni column purification, (A) is the SDS-PAGE gel image of V1SA-3005Hz6 and 3005Hz6-V1SA, (B) is the SDS-PAGE gel image of V1DP-3005Hz6, wherein the supernatant of the cell culture medium after transient transfection of each protein is marked as Input, the flow-through that cannot bind to the Ni column is marked as FT, and the eluted purification product is marked as E.

[0166] Figure 10: Fusion construct of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody, and ELISA activity detection of humanized antibody 3005Hz6 binding to HSA.

[0167] Figure 11: VEGFR2 competition ELISA activity test of fusion constructs and monovalent Nanobody V1-SA1.

[0168] Figure 12: SDS-PAGE image of the fusion construct of humanized antibody 3005Hz6 and bivalent anti-VEGFA nanobody after purification, (A) is a gel stained image of V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, wherein the flow-through is marked as FT, the elution products of V1SA-3005Hz6-V1SA are marked as A1 and A2 components, and the elution products of 3005Hz6-2V1SA are marked as A5 and A6 components, (B) is a gel stained image of the unlabeled 3005Hz6-2V1SA protein product, (C) is a gel stained image of V1DP-3005Hz6-V1DP, and (D) is a gel stained image of 3005Hz6-2V1DP.

[0169] Figure 13: ELISA test of the fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP binding to HSA.

[0170] Figure 14: The affinity of the protein to HSA was tested using the SPR method, wherein Figure (A) shows the binding and dissociation curves of the fusion construct V1SA-3005Hz6-V1SA and HSA, Figure (B) shows the binding and dissociation curves of the fusion construct 3005Hz6-2V1SA and HSA, and Figure (C) shows the binding and dissociation curves of the antibody 3005Hz6 and HSA.

[0171] Figure 15: The affinity of the protein to VEGFA was tested using the SPR method, wherein Figure (A) shows the binding and dissociation curves of V1SA-3005Hz6-V1SA and VEGFA, Figure (B) shows the binding and dissociation curves of 3005Hz6-2V1SA and VEGFA, and Figure (C) shows the binding and dissociation curves of the positive control aflibercept and VEGFA.

[0172] Figure 16: Fusion construct inhibits VEGFA-induced HUVEC proliferation experiment, wherein Figure (A) shows the test results of V1SA-3005Hz6-V1SA, and the premixture of V1SA-3005Hz6-V1SA and HSA (V1SA-3005Hz6-V1SA+HSA), and Figure (B) shows the test results of 3005Hz6-2V1SA, and the premixture of 3005Hz6-2V1SA and HSA (3005Hz6-2V1SA+HSA).

[0173] Figure 17: Fusion construct inhibits VEGFA-induced reporter gene cell experiment, wherein Figure (A) shows the test results of V1SA-3005Hz6-V1SA and V1SA-3005Hz6-V1SA+HSA, and Figure (B) shows the test results of 3005Hz6-2V1SA and 3005Hz6-2V1SA+HSA. DETAILED DESCRIPTION

[0174] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0175] Example 1 Alpaca immunization and antibody library construction

[0176] 1. Alpaca immunity and potency testing

[0177] Alpacas were immunized with emulsified human serum albumin (HSA) (purchased from Sigma-Aldrich, Catalog No. A3782). Each immunization was administered with 1 mg of HSA, once every two weeks, for a total of three immunizations. Serum was collected one week after the second immunization for titer determination.

[0178] Serum titer detection method is:

[0179] 1) Dilute HSA antigen to 1 μg / mL with CBS buffer, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight;

[0180] 2) Wash the plate three times with PBST (PBS containing 0.1% Tween 20, pH = 7.4). Add 200 μL of skim milk powder to each well and let it stand at room temperature for 1 hour. Wash the plate three times with PBST.

[0181] 3) Sample addition: Dilute the immunized serum and negative serum 1000-fold, 3000-fold, 9000-fold, 27000-fold, 81000-fold, and 243000-fold, respectively. Add 100 μL of each to the sealed wells, incubate at room temperature for 1 hour, and wash the plate three times with PBST.

[0182] 4) Secondary antibody: Add 100 μL of HRP-labeled Goat Anti-Alpaca IgG to each well, incubate at room temperature for 1 hour, and wash the plate three times with PBST.

[0183] 5) TMB color development: Add 100 μL / well of TMB color development solution to the ELISA plate and develop for 15 min. Add 50 μL / well of stop solution, place the plate in a microplate reader, and read the absorbance at 450 nm (OD450).

[0184] The serum titer test after the second and third alpaca immunization is shown in Table 1. It can be seen that after the serum was diluted 243,000 times, the signal value of binding to human serum albumin was above 0.59, which met the library construction standard. The peripheral blood of the alpaca after the third immunization was used for phage library construction.

[0185] Table 1 Serum titer detection after the second and third albumin alpaca immunization

[0186] 2. Phage library construction

[0187] Peripheral blood was drawn from alpacas, and total RNA was extracted from the isolated alpaca PBMC using Trizol. Reverse transcription of cDNA was performed, and the variable region VH fragment (approximately 700 bp in length) was amplified twice using nanobody-specific primers. The resulting fragment and the pcomb3X vector were digested with SfiI enzyme, mixed in appropriate proportions, and ligated with T4 ligase. After ligation, the fragments were used for electroporation into XL1-Blue competent cells. Based on the colony growth of the dilution of the competent cells on the resistance-containing plate, the transformation library capacity of the alpaca was calculated to be 6.24×10 8 .

[0188] Example 2 Screening of Antibody Library to Obtain Antibodies Cross-binding to Human, Mouse, and Cynomolgus Monkey Albumin

[0189] 1. Phage library screening

[0190] The screening process is:

[0191] 1) Coat the plate with antigens human serum albumin (HSA) and mouse albumin (MSA) (purchased from Sigma-Aldrich, Cat. No. A3559). Simultaneously, set up a negative control (coat the plate with 3% skim milk powder). Incubate at room temperature for 1 hour. Discard the blocking buffer and wash with PBST.

[0192] 2) Add about 5×10 12 The pfu library was incubated with antigen on antigen plates at 37°C for 2 hours, and the plates were washed with 0.1% PBST.

[0193] 3) Elution was performed with low-pH glycine-HCl, followed by neutralization with Tris-HCl until the pH reached 7.4.

[0194] 4) The eluate was mixed with E. coli XL1-Blue and incubated at 37°C with shaking.

[0195] 5) After adding phage, culture at 30°C overnight and collect the supernatant.

[0196] 6) Repeat 1)-5) to obtain the second round library.

[0197] 7) Antigen binding ELISA tests were performed on the second-round amplified library obtained under MSA-coating conditions. In these ELISA experiments, HSA and MSA were coated separately using CBS buffer. The ELISA test results are shown in Table 2. As can be seen, phages with strong binding to HSA and MSA were also obtained under this enrichment condition.

[0198] Table 2 Detection results of the second round of phage library obtained by enrichment under MSA coating conditions combined with HSA and MSA

[0199] Summary: Antigen binding ELISA of the second-round phage library obtained by MSA enrichment showed positive binding to both HSA and MSA, indicating that phages binding to HSA and MSA were enriched, and binding screening at the phage monoclonal level can be carried out.

[0200] 2. Phage monoclonal screening and sequencing

[0201] The experimental process is:

[0202] 1) 96 monoclonal colonies were selected from the second-round phage library enriched by coating with MSA for phage expression and coated with HSA, MSA, and monkey albumin for ELISA binding activity detection.

[0203] 2) Coating antigens: HSA, MSA, and monkey albumin. The protein concentration during coating is 0.5 μg / mL. Coating is carried out at 4°C overnight.

[0204] 3) Blocking: 3% milk powder, 200 μL / well, room temperature for 1 hour.

[0205] 4) Add 10-fold diluted phage expression supernatant, 100 μL / well, and incubate at room temperature for 1 hour.

[0206] 5) Add detection antibody Anti-M13 Antibody (HRP labeled) (concentration: 0.2 μg / mL), 100 μL / well, and incubate at room temperature for 1 hour.

[0207] 6) Add 200 μL / well of TMB colorimetric solution, develop for 20 minutes, then add 50 μL / well of stop solution. Measure OD450 using a microplate reader.

[0208] From the 96 phage clones in the second round of phage library enriched by MSA coating, clone number 3005 was selected. The ELISA test results are shown in Table 3.

[0209] Table 3 Activity detection of phage monoclonal 3005 binding to HSA, MSA and monkey albumin

[0210] 7) After DNA sequencing and codon translation, the amino acid sequence of the candidate Nanobody is obtained. Among them, the amino acid sequence of the CDR region (CDR-H1, CDR-H2, CDR-H3) of the preferred anti-albumin Nanobody 3005 is shown in Table 4, the nucleic acid sequence of the CDR region is shown in Table 5, the amino acid sequence of the antibody heavy chain variable region (VH) is shown in Table 6, and the nucleic acid sequence is shown in Table 7.

[0211] Table 4 CDR region amino acid sequence of anti-albumin nanobody 3005

[0212] Table 5 Nucleic acid sequences encoding the CDR regions of anti-albumin nanobody 3005

[0213] Table 6 Anti-albumin Nanobody 3005 amino acid sequence

[0214] Table 7 Nucleic acid sequence of anti-albumin nanobody 3005

[0215] Example 3 Detection of the activity of anti-albumin candidate antibodies binding to albumins from different species

[0216] 1. Expression of anti-albumin candidate antibodies

[0217] The candidate antibody is expressed in mammalian cells, and after protein purification, the antigen binding activity test is performed to verify the activity of the candidate antibody in binding to albumin at the protein level.

[0218] By gene synthesis, expression plasmids of His-tagged antibody 3005 and positive control antibody Alb Nb were constructed. The positive control antibody Alb Nb is a nanobody against human serum albumin, and the antibody amino acid sequence is derived from SEQ ID NO: 62 in patent US2007 / 0269422 A1.

[0219] The tPA secretion signal peptide (MDAMKRGLCCVLLLCGAVFVSPS) (SEQ ID NO: 36) was added to the N-terminus of the above-mentioned antibody. The linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The gene sequence was ligated into the expression vector pCDNA3.1(+) via the restriction enzyme cleavage sites NheI and XbaI. After plasmid sequencing was confirmed, the plasmid was extracted in an endotoxin-free manner, transiently expressed in suspension 293F cells, and affinity purified using a Ni column.

[0220] Figures 1(A) and 1(B) show SDS-PAGE results of His-tagged antibodies Alb Nb and 3005 purified on a Ni column, demonstrating molecular weights consistent with expectations (~15 kDa). In Figure 1(A), the supernatant from the cell culture medium after transient transfection with antibody Alb Nb is labeled Input, the flow-through that failed to bind to the Ni column is labeled FT, and the antibody Alb Nb that bound to the Ni column and was eluted is labeled E.

[0221] At the same time, a fusion protein of antibody 3005 and the positive control antibody Alb Nb with Fc was constructed. The tPA secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) was added to the N-terminus of the antibody coding sequence through primer design. The amino acid sequence of human IgG1 Fc was added to the C-terminus of the antibody sequence through homologous recombination:

[0222] Amino acid sequence of IgG1 Fc:

[0223] The anti-albumin nanobody-Fc construct gene fragment was inserted into the mammalian cell expression vector pCDNA3.1(+). The amino acid sequence of the anti-albumin nanobody-Fc fusion protein is shown in Table 8, and the nucleic acid sequence is shown in Table 9.

[0224] Table 8 Amino acid sequence of anti-albumin nanobody-Fc fusion protein

[0225] Table 9 Nucleic acid sequences encoding anti-albumin nanobody-Fc fusion proteins

[0226] After the expression plasmid of the Fc fusion protein is sequenced correctly, a large amount of endotoxin-free extraction is performed, and transient expression in suspension 293F cells and Protein A affinity purification are carried out to obtain the target protein.

[0227] 2. Detection of albumin binding activity of candidate anti-albumin antibodies

[0228] Bio-layer interferometry (BLI) was used to test the affinity of monovalent nanobodies for binding to albumins from different species. Eight NTA sensors were used to bind to the same his-tag monovalent nanobody (at a concentration of 1 μg / mL). The antigens were human serum albumin (HSA) (Baxter AG), mouse albumin (MSA) (purchased from Equitech-bio, Catalog No. MSA62-1000), and rat albumin (purchased from abcam, Catalog No. ab198656). The BLI assay was performed at a gradient of antigen concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM. The binding time was 180 seconds, and the dissociation time was 360 seconds.

[0229] Table 10 shows the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of the monovalent nanobody 3005 with his tag binding to albumin from different species after "1:1" model fitting. D ).

[0230] Table 10 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of Nanobody 3005 binding to albumin from different species D )

[0231] The data showed that 3005 binds to HSA and MSA with high affinity, and also binds to rat albumin.

[0232] The ELISA method was used to test the activity of 3005 binding to albumin from different species. The experimental method was as follows:

[0233] Dilute the antigen in ELISA coating buffer to a final concentration of 1 μg / mL for human serum albumin (HSA) (Baxter AG), 2 μg / mL for mouse albumin (MSA) (Equitech-bio, Catalog No. MSA62-1000), and 2 μg / mL for rat albumin (abcam, Catalog No. ab198656). Add 100 μL of the antigen dilution to the ELISA plate and coat overnight at 4°C. After blocking with 5% skim milk powder, the plates were diluted with PBST (0.1% Tween 20 in PBS, pH 7.4) and serial dilutions of the candidate antibody 3005-Fc and the positive control antibody Alb Nb-Fc (0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM). The plates were incubated at 37°C for 1 hour. After washing, HRP-conjugated goat anti-human IgG Fc antibody (Abbkine, Catalog No. A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0234] The ELISA test results of antibodies 3005-Fc and Alb Nb-Fc binding to albumins from different species are shown in FIG2 , and the EC50 of the binding ELISA experiments are summarized in Table 11 .

[0235] Table 11 Summary of EC50 of ELISA experiments of antibody 3005-Fc and positive control Alb Nb-Fc binding to albumin from different species

[0236] The results showed that antibody 3005-Fc efficiently bound to HSA and MSA, with binding activity similar to that of the positive control antibody Alb Nb-Fc. While the positive control antibody Alb Nb-Fc had very weak binding activity to rat serum albumin, antibody 3005-Fc had strong binding activity to rat serum albumin.

[0237] Example 4 Humanization of anti-albumin candidate antibodies

[0238] 1. Humanization transformation, expression and purification of candidate antibodies

[0239] Humanization was performed by transplanting the CDR region of the antibody. Using candidate Nanobody 3005 (SEQ ID NO: 4) as the starting antibody, humanization of the antibody was performed. First, the homology between the candidate antibody 3005 sequence and the human antibody sequence was compared using the antibody databases IGBLAST and IMGT. The humanized antibody was then constructed by combining the human antibody framework sequence with the three CDR region sequences of candidate antibody 3005 (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3).

[0240] A secretion signal peptide was added to the antibody's N-terminus, and the linkers GGGGS (SEQ ID NO: 37) and 6xHis were added to the antibody's C-terminus. The coding DNA sequence was synthesized and ligated into the expression vector pCDNA3.1(+) via the restriction enzyme sites NheI and XbaI. The plasmid was sequenced correctly and extracted in an endotoxin-free bulk format. Transient expression was performed in suspension 293F cells and affinity purified using a Ni column.

[0241] The amino acid sequence of the humanized modified antibody of Nanobody 3005 is shown in Table 12, and the nucleic acid sequence is shown in Table 13.

[0242] Table 12 Amino acid sequence of 3005 humanized nanobody

[0243] Table 13 Nucleic acid sequences encoding 3005 humanized nanobody

[0244] The His-tagged humanized protein was purified by Ni column and separated by SDS-PAGE electrophoresis. The molecular weight was consistent with the expected value (~13 kDa). The staining results are shown in Figure 3.

[0245] 2. Binding activity detection of humanized antibodies

[0246] In this example, the ELISA method was used to detect the binding activity of the original candidate antibody 3005 and the humanized antibody to mouse albumin MSA.

[0247] In the experiment, mouse albumin MSA (coating protein concentration of 5 μg / mL) was coated in an ELISA plate at 100 μL / well at 4°C overnight. After blocking with 5% skim milk powder, dilutions of the His-tagged protein to be tested (0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM) were prepared in PBST (PBS containing 0.1% Tween 20, pH = 7.4). The proteins tested were the original antibody 3005 and four humanized antibodies. The protein dilutions were added to the ELISA plate and incubated at 37°C for 1 hour. After washing, HRP-conjugated mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0248] The ELISA test results of antibody 3005 and its humanized antibody binding to MSA are shown in Figure 4. The EC50 of the binding ELISA experiments is summarized in Table 14.

[0249] Table 14 Summary of EC50 and maximum binding values ​​of ELISA experiments of antibody 3005 and its humanized antibodies binding to MSA

[0250] The results showed that the humanized antibody 3005Hz6 had similar MSA binding activity to the original antibody 3005. The MSA binding activity of other humanized antibodies was worse than that of 3005Hz6.

[0251] The ELISA method was used to detect the binding activity of the original candidate antibody 3005 and the humanized 3005Hz6 to HSA and rat albumin.

[0252] In the experiment, HSA or rat albumin (coating protein concentration of 2 μg / mL) was coated in an ELISA plate at 100 μL / well overnight at 4°C. After blocking with 5% skim milk powder, dilutions of the His-tagged protein to be tested (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 10, and 100 nM) were prepared in PBST (PBS containing 0.1% Tween 20, pH = 7.4). The tested proteins included the positive control antibody Alb Nb, the original antibody 3005, and the humanized antibody 3005Hz6. The protein dilutions were added to the ELISA plate and incubated at 37°C for 1 hour. After washing, HRP-conjugated mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0253] The ELISA results are shown in Figures 5(A) and 5(B). The EC50 values ​​of the binding ELISA curve fitting of Figure 5 are shown in Table 15.

[0254] Table 15 Summary of EC50 of antibody 3005 and its humanized antibody 3005Hz6 binding to HSA and rat albumin

[0255] As can be seen from Figure 5 and Table 15, the binding activity of humanized antibody 3005Hz6 to HSA and rat albumin is comparable to that of original antibody 3005, and both are stronger than the positive control antibody Alb Nb.

[0256] The surface plasmon resonance (SPR) method was used to detect the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 binding to HSA and MSA. D). In the experiment, a Biacore 8K instrument (Cytiva) was used, and the Series S Sensor Chip NTA was used to capture the His-tagged humanized antibody 3005Hz6. The analytes were HSA or MSA in gradient concentrations, and the analytes were diluted with mobile phase buffer (components: 10mM HEPES, 150mM NaCl, 0.05% v / v Tween-20, pH=7.4). The binding time was 150 seconds, and the dissociation time was 900 seconds. The binding and dissociation curves of antibody 3005Hz6 with HSA are shown in Figure 6 (A), and the binding and dissociation curves of antibody 3005Hz6 with MSA are shown in Figure 6 (B). The "Two state reaction" model was used to fit the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of 3005Hz6 binding to HSA and MSA. D ), the results are shown in Table 16.

[0257] Table 16 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 with HSA and MSA D )

[0258] As shown in Table 16, the affinity of humanized antibody 3005Hz6 for binding to HSA and MSA is similar, and the K values ​​determined by SPR are D About 11nM.

[0259] In combination with Table 10 of Example 3, the K values ​​of the original antibody 3005 with HSA and MSA were determined by BLI method. D The value is about 5nM, which is consistent with the K D The difference is within 3 times, so it is believed that the affinity of humanized antibody 3005Hz6 for binding to HSA and MSA is similar to that of original antibody 3005.

[0260] Example 5 Risk Site Modification of Humanized Antibody 3005Hz6 and Activity Testing

[0261] 1. Risk site mutation of humanized antibody 3005Hz6 and its expression and purification

[0262] Sequence analysis of the 3005Hz6 antibody revealed that its CDR-H2 region (SEQ ID NO: 2) contains the amino acid motif DG, which is susceptible to isomerization of aspartic acid D. Therefore, the DG amino acid motif in the CDR-H2 region of 3005Hz6 was modified to DA, DS, or EG, respectively, to reduce the risk of changes in protein structure and activity. The names of the modified antibodies and their CDR region amino acid sequences are shown in Table 17, and their nucleic acid sequences are shown in Table 18. The amino acid sequence of the antibody heavy chain variable region (VH) is shown in Table 19, and its nucleic acid sequence is shown in Table 20.

[0263] Table 17 CDR region amino acid sequences of humanized antibody 3005Hz6 mutants

[0264] Table 18 Nucleic acid sequences encoding the CDR regions of humanized antibody 3005Hz6 mutants

[0265] Table 19 Amino acid sequences of humanized Nanobody 3005Hz6 mutants

[0266] Table 20 Nucleic acid sequences encoding humanized Nanobody 3005Hz6 mutants

[0267] A secretion signal peptide was added to the N-terminus of the antibody 3005Hz6 mutant, and the linkers GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus. The coding DNA sequence was obtained by gene synthesis (see Table 20) and then ligated into the expression vector pCDNA3.1(+) via the restriction enzyme cleavage sites NheI and XhoI. The plasmid was sequenced correctly and extracted in an endotoxin-free manner. Transient expression was performed in suspended 293F cells. The cell culture supernatant was harvested and purified using a Ni column affinity purification. The SDS-PAGE results of the purified products are shown in Figure 7(A). The culture supernatant of cells transiently expressing 3005Hz6(EG) and 3005Hz6(DS) is labeled as Input, the flow-through that did not bind to the Ni column is labeled as FT, and the eluted purified product is labeled as E. The supernatant of cells transiently expressing 3005Hz6(DA) protein was purified using a Ni column and then by cation exchange purification. The SDS-PAGE results of the purified protein are shown in Figure 7(B). The molecular weight of the three mutant proteins was 13 kDa.

[0268] 2. Activity test of humanized antibody 3005Hz6 mutant

[0269] ELISA assays were performed to assess HSA binding activity of the humanized 3005Hz6 antibody mutants, 3005Hz6(DA), 3005Hz6(DS), and 3005Hz6(EG). In the ELISA assay, 2 μg / mL HSA was coated and a concentration gradient of 3005Hz6 or its mutants was added. HSA binding was measured using an HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005). The ELISA results are shown in Figure 8 , and the EC50 values ​​are shown in Table 21.

[0270] Table 21 Summary of EC50 of humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) binding to HSA

[0271] As can be seen from Table 21, the humanized antibody 3005Hz6 and its mutant proteins have similar HSA binding activities.

[0272] Example 6 Fusion construct of monovalent anti-VEGFA nanobody and humanized anti-albumin antibody

[0273] 1. Design of a fusion construct of monovalent anti-VEGFA nanobody and humanized anti-albumin antibody 3005Hz6

[0274] Using anti-VEGFA nanobody as the effector molecule, a fusion construct of anti-VEGFA antibody and anti-albumin antibody 3005Hz6 was constructed.

[0275] The screening technology scheme for anti-VEGFA nanobodies in this embodiment can be referred to the patent application number PCT / CN2023 / 118600, which is briefly summarized as follows:

[0276] The anti-VEGFA nanobody sequences used in this example were screened from a human VEGFA-immunized alpaca library. The phage library construction and screening process was as follows: Human VEGFA165 (untagged, purchased from Sino Biological, Cat. No. HPLC-10008-HNAH, hereinafter referred to as VEGFA) was emulsified and immunized against alpacas to construct a phage display library. After four rounds of enrichment screening, single clones were selected from a 96-well plate and tested for phage binding activity to VEGFA. Phage with VEGFA binding activity were sequenced and codon translated to obtain the amino acid sequences of candidate nanobodies. The amino acid sequence of the CDR region of the preferred anti-VEGFA nanobody V1 is shown in Table 22, and the nucleic acid sequence is shown in Table 23. The amino acid sequences of Nanobody V1 and its nine humanized antibodies are shown in Table 24, and the nucleic acid sequences are shown in Table 25.

[0277] Table 22 CDR region amino acid sequence of anti-VEGFA nanobody V1

[0278] Table 23 Nucleic acid sequences encoding the CDR regions of anti-VEGFA nanobody V1

[0279] Table 24 Amino acid sequences of anti-VEGFA nanobody V1 and its humanized antibodies

[0280] Table 25 Nucleic acid sequences encoding anti-VEGFA nanobody V1 and its humanized antibodies

[0281] Antibodies with high thermal stability are more conducive to antibody production and long-term storage. Therefore, the thermal stability Tm values ​​(melting temperature) of different anti-VEGFA nanobodies were compared. The Tm values ​​of anti-VEGFA nanobody V1 and nine humanized antibodies were measured using the UNCLE instrument (Unchained Labs). The results are shown in Table 26.

[0282] Table 26 Thermal stability Tm values ​​of anti-VEGFA nanobody V1 and its 9 humanized antibodies

[0283] As can be seen from Table 26, the Tm values ​​of V1-DP and V1-SA1 are relatively high, indicating that the thermal stability of these two antibodies is likely to be better than that of other humanized antibodies.

[0284] Humanized anti-VEGFA nanobodies with higher melting temperature Tm values: V1-SA1 (SEQ ID NO: 20) and V1-DP (SEQ ID NO: 21) were selected for the design of fusion constructs with humanized anti-albumin antibody 3005Hz6. According to different tandem sequences, a total of three fusion constructs with GGS as the linker were designed. The amino acid sequences are shown in Table 27, and the nucleic acid sequences are shown in Table 28.

[0285] Table 27 Amino acid sequences of fusion constructs of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0286] Table 28 Nucleic acid sequences encoding fusion constructs of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0287] A secretion signal peptide was added to the N-terminus of the fusion construct, and the linkers GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence was obtained by gene synthesis (see Table 28) and then ligated into the expression vector pCDNA3.1(+) via the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. Transient expression was performed using suspended 293F cells, and the cell culture supernatant was harvested and purified using a Ni column affinity purification. The SDS-PAGE results of the purified products are shown in Figure 9, where the supernatant of the cell culture medium after transient transfection of each protein is labeled as Input, the flow-through that cannot bind to the Ni column is labeled as FT, and the eluted purified product is labeled as E. The molecular weight of the target protein is approximately 28 kDa.

[0288] 2. Activity detection of the fusion construct of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0289] This example tests whether the two Nanobody elements constituting the fusion construct maintain their original activity.

[0290] The HSA binding activity of the anti-albumin nanobody 3005Hz6 in the fusion construct was tested using the same method as in "2. Activity testing of humanized antibodies" in Example 4. The ELISA results are shown in Figure 10, and the EC50 values ​​are shown in Table 29.

[0291] Table 29 Summary of EC50 of fusion constructs or 3005Hz6 binding to HSA

[0292] As shown in Table 29, the HSA binding activity of the fusion construct is similar to that of 3005Hz6 alone, indicating that the fusion of 3005Hz6 with other antibody fragments does not affect the HSA binding activity of 3005Hz6. Furthermore, the anti-VEGFA antibody can be fused to the N- or C-terminus of the anti-albumin antibody; the different positions do not affect the albumin binding activity of the fusion protein.

[0293] The activity of the anti-VEGFA nanobody in the fusion construct was detected using a VEGFR2 competition ELISA method, and the activity of the fusion construct was compared with that of the VEGFA antibody alone.

[0294] Specifically, the ELISA plate was coated with goat anti-human IgG Fc protein (purchased from Solarbio, catalog number: SPA105) at a coating concentration of 5 μg / mL at 4°C overnight. After washing with PBST and blocking with milk powder, VEGFR2 extracellular domain-human IgG1 Fc fusion protein (VEGFR2-Fc, prepared in-house) was added. The amino acid sequence of VEGFR2-Fc is:

[0295] A concentration gradient of the fusion construct was incubated with a constant final concentration (0.4 nM) of biotinylated VEGFA165-Avi-His for 1 hour. VEGFA165-Avi-His was prepared in-house by adding a linker, an Avi tag, and a 6xHis tag to the C-terminus of human VEGFA165. The VEGFA165-Avi-His expression plasmid was co-transfected with a BirA enzyme expression plasmid into 293F cells. During protein expression, the Avi tag was biotinylated by BirA enzyme. The amino acid sequence of VEGFA165-Avi-His is:

[0296] The mixture of the fusion construct and VEGFA165-Avi-His was added to the ELISA plate with captured VEGFR2-Fc and incubated at 37°C for 1 hour. The biotin signal of VEGFA165-Avi-His was detected using HRP-labeled streptavidin-HRP (purchased from Sangon Biotechnology, catalog number: D111054-0001). The plate was incubated at room temperature for 45 minutes. After washing, 100 μL / well of TMB colorimetric solution was added to the ELISA plate and color was developed for 15 minutes. 50 μL / well of stop solution was added, and the plate was placed in a microplate reader to read the absorbance at 450 nm (OD450).

[0297] The competitive VEGFR2 activity of the fusion construct and the monovalent Nanobody V1-SA1 is shown in Figure 11, and the IC50 of the VEGFR2 competition experiment is summarized in Table 30.

[0298] Table 30 Summary of IC50 of fusion constructs and monovalent Nanobody V1-SA1 competing for VEGFR2

[0299] As can be seen from Table 30, the VEGFR2 competitive activity of the three fusion constructs composed of 3005Hz6 and V1-SA1 or V1-DP in series is similar to that of the monovalent nanoantibody V1-SA1 (IC50 ratio is 0.81-1.08), indicating that the fusion of anti-VEGFA antibody with 3005Hz6 does not affect the anti-VEGFA activity. Moreover, the anti-VEGFA antibody can be fused to the N-terminus or C-terminus of the anti-albumin antibody without affecting the activity of the anti-VEGFA antibody in the fusion construct.

[0300] The data in Tables 29 and 30 show that the fusion construct of the monovalent anti-VEGFA Nanobody (V1-SA1 or V1-DP) and the anti-albumin Nanobody (3005Hz6) has strong competitive activity for VEGFR2 and binding to HSA. This indicates that in the fusion construct, the antibody elements that bind to different antigens can function independently, and there is little mutual interference between the antibody elements. Therefore, antibodies that bind to other antigens can be fused with the humanized anti-albumin antibody 3005Hz6 to construct a multifunctional fusion construct.

[0301] Example 7 Fusion construct of bivalent anti-VEGFA nanobody and humanized anti-albumin antibody

[0302] 1. Design of a fusion construct of a bivalent anti-VEGFA nanobody and an anti-albumin nanobody

[0303] Since VEGFA is a homodimer in vivo, to increase the efficiency of nanoantibodies binding to VEGFA and to enhance the efficiency of nanoantibodies in blocking VEGFA / VEGFR interactions, anti-VEGFA nanoantibodies are in a bivalent form, i.e., two nanoantibodies with the same sequence are linked in series. In order to increase the molecular weight of the bivalent anti-VEGFA nanoantibody in vivo and extend the half-life of the bivalent antibody by binding to albumin, the bivalent anti-VEGFA nanoantibody was fused with the humanized anti-albumin antibody 3005Hz6 for expression. The amino acid sequence of the humanized antibody 3005Hz6 and the bivalent anti-VEGFA nanoantibody V1-SA1 or V1-DP fusion construct is shown in Table 31, and the nucleic acid sequence is shown in Table 32.

[0304] Table 31 Amino acid sequences of fusion constructs of bivalent anti-VEGFA nanobodies and humanized anti-albumin antibody 3005Hz6

[0305] Table 32 Nucleic acid sequences encoding fusion constructs of bivalent anti-VEGFA nanobodies and humanized anti-albumin antibody 3005Hz6

[0306] The secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) was added to the N-terminus of the fusion construct, and the linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence (see Table 32) was obtained by gene synthesis and then ligated into the expression vector pCDNA3.1(+) via the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in an endotoxin-free manner. Transient expression was performed in suspended 293F cells, and the cell culture supernatant was harvested and affinity purified using a Ni column. For the fusion construct 3005Hz6-2V1SA (SEQ ID NO: 33), an untagged protein was also constructed. After adding the secretion signal peptide to the N-terminus of the protein, it was ligated into the expression vector and transiently expressed in 293F cells.

[0307] After Ni column purification, V1SA-3005Hz6-V1SA (SEQ ID NO: 32) and 3005Hz6-2V1SA (SEQ ID NO: 33) were subjected to cation exchange purification. The purified product fractions were subjected to SDS-PAGE. The results are shown in Figure 12(A). The flowthrough is labeled FT, the eluted products of V1SA-3005Hz6-V1SA are labeled fractions A1 and A2, and the eluted products of 3005Hz6-2V1SA are labeled fractions A5 and A6. Cation exchange purification of untagged 3005Hz6-2V1SA was performed, and the purified product staining is shown in Figure 12(B). V1DP-3005Hz6-V1DP and 3005Hz6-2V1DP were purified by Ni column, and the purified product staining is shown in Figure 12(C). The molecular weight of the fusion construct is approximately 41 kDa.

[0308] 2. ELISA testing and association and dissociation rate assay (SPR) of fusion constructs

[0309] The HSA-binding activity of the fusion constructs V1SA-3005Hz6-V1SA (SEQ ID NO: 32), 3005Hz6-2V1SA (SEQ ID NO: 33), and 3005Hz6-2V1DP (SEQ ID NO: 35) was assessed by ELISA. Human serum albumin (HSA) (Baxter AG) was diluted to 2 μg / mL and added to an ELISA plate at 100 μL / well for overnight coating at 4°C. After blocking with 5% skim milk powder and washing with PBST (PBS containing 0.1% Tween 20, pH 7.4), serial dilutions of the His-tag fusion constructs were added to the plate at 100 μL / well for incubation at 37°C for 1 hour. After washing with PBST, HRP-conjugated mouse anti-His tag monoclonal antibody (Proteintech, Cat. No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader (purchased from Thermo Scientific, Multiskan SkyHigh, Catalog No. A51119700C).

[0310] The ELISA results are shown in Figure 13, and the EC50 values ​​are shown in Table 33.

[0311] Table 33 Summary of EC50 of fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP binding to HSA

[0312] As can be seen from Table 33, the fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP all have strong HSA binding activity, indicating that the antibody 3005Hz6 in the fusion construct can exert complete biological activity.

[0313] The affinity of the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA to HSA was determined by surface plasmon resonance (SPR) method.

[0314] The experiments were performed using a Biacore 8K instrument (Cytiva) and a Series S Sensor Chip NTA to capture the His-tagged anti-albumin antibody 3005Hz6 or the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, respectively. The analyte was a gradient of HSA concentrations diluted in a mobile phase buffer (10 mM HEPES, 150 mM NaCl, 0.05% v / v Tween-20, pH 7.4). The binding time between the ligand and the analyte was 150 seconds, and the dissociation time was 900 seconds. The binding and dissociation curves for the ligand and analyte are shown in Figure 14.

[0315] The “1:1 binding” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of 3005Hz6, V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA with HSA. D ), the results are shown in Table 34.

[0316] Table 34 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of anti-albumin antibody 3005Hz6 and fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA to HSA D )

[0317] As shown in Table 34, the fusion construct 3005Hz6-2V1SA has a high affinity with HSA, K D Similar to the K of antibody 3005Hz6 and HSA D (K D is 12.7-35.8nM).

[0318] 3. Binding and dissociation rate determination of fusion constructs with VEGFA (SPR)

[0319] The affinity of the fusion constructs V1SA-3005Hz6-V1SA (His-tagged), 3005Hz6-2V1SA (untagged), and the positive control aflibercept for VEGFA was determined using SPR. SPR experiments were performed using a Biacore 8K (Cytiva). Biotinylated VEGFA165-Avi-His (SEQ ID NO: 40) was captured using a Series S Sensor Chip CAP at a capture level of approximately 40 RU. Each protein was diluted in the mobile phase buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20) and a two-fold protein concentration gradient was established. Kinetic analysis was performed with an association time of 150 seconds and a dissociation time of 900 seconds. The flow rate was 30 μL / min.

[0320] The binding and dissociation curves of V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and the positive control aflibercept with VEGFA are shown in Figure 15(A), Figure 15(B) and Figure 15(C), respectively. The binding and dissociation curves were fitted with the "1:1 binding" model to obtain the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ), the results are shown in Table 35.

[0321] Table 35 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and positive control aflibercept to VEGFA D )

[0322] As can be seen from Table 35, under the SPR experimental conditions of capturing biotin-modified VEGFA165-Avi-His, the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA have extremely high binding affinity to VEGFA (KD = 2.4-5.4pM). Under the same experimental conditions, the affinity of the positive control drug aflibercept to VEGFA is one order of magnitude lower than that of V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, with a KD of approximately 110pM.

[0323] Example 8 Study on the inhibition of VEGFA-stimulated HUVEC proliferation activity by fusion constructs

[0324] In this example, primary human umbilical vein endothelial cells (HUVEC) were used to test whether the fusion construct could block VEGFA-stimulated HUVEC proliferation at the cellular level.

[0325] Since the fusion construct binds to HSA in the human body, in this experiment, the difference in the inhibitory activity of the fusion construct with or without pre-incubation with HSA in terms of HUVEC proliferation was compared.

[0326] In the experiment, a human VEGFA165 solution (purchased from GenScript, catalog number: Z03073) at a concentration of 210 ng / mL was prepared using endothelial cell culture medium (ECM) containing 0.5% FBS (purchased from Zhongqiao Xinzhou, catalog number: ZQ-1304). A gradient dilution of the fusion construct 3005Hz6-2V1SA with or without the addition of HSA at the same molar concentration (labeled as: 3005Hz6-2V1SA+HSA and 3005Hz6-2V1SA, respectively) and a gradient dilution of the fusion construct V1SA-3005Hz6-V1SA with or without the addition of HSA at the same molar concentration (labeled as: V1SA-3005Hz6-V1SA+HSA and V1SA-3005Hz6-V1SA, respectively) were prepared. The positive control was the marketed drug aflibercept (Bayer) at the same molar concentration gradient. In a 96-well plate, 50 μL of a gradient of test protein concentrations (3005Hz6-2V1SA+HSA, 3005Hz6-2V1SA, V1SA-3005Hz6-V1SA+HSA, V1SA-3005Hz6-V1SA, and aflibercept) were mixed with 50 μL of VEGFA165 solution. A control without VEGFA165 (expected to result in the slowest proliferation) and a control with VEGFA165 alone (expected to result in the fastest proliferation) were set up. The test protein and VEGFA165 mixture was incubated in a 37°C, 5% CO2 incubator for 1.5-2 hours. Primary HUVEC cells (purchased from the National Stem Cell Transformation Resource Center, catalog number: DFSC-EC-01) were digested and centrifuged, and then resuspended in ECM containing 0.5% FBS. 50 μL of cell suspension was added to a 96-well plate pre-mixed with the test protein and VEGFA165, and 1.2×10 4Cells were plated at a final concentration of 70 ng / mL for VEGFA165. The cells were cultured in a 37°C, 5% CO2 incubator for 68-72 hours. After incubation, 16 μL of CCK-8 staining solution (available from Solarbio, Catalog No. CA-1210) was added to each well and incubated in a 37°C incubator for 2.5-4 hours. HUVEC cell proliferation activity was measured by reading absorbance at 450 nm using a microplate reader. IC50 values ​​were calculated using four-parameter nonlinear regression analysis using GraphPad Prism 8 software.

[0327] Calculate the inhibition rate of the test protein on cell proliferation at different concentrations. The calculation formula of the inhibition rate is:

[0328] The experimental results of the inhibition of VEGFA-stimulated HUVEC proliferation by the fusion constructs V1SA-3005Hz6-V1SA (with or without pre-incubation of HSA) and 3005Hz6-2V1SA (with or without pre-incubation of HSA) are shown in Figures 16(A) and 16(B), respectively.

[0329] The IC50 and maximum inhibition rate of the fusion construct V1SA-3005Hz6-V1SA in inhibiting HUVEC proliferation are shown in Table 36, and the IC50 and maximum inhibition rate of the fusion construct 3005Hz6-2V1SA in inhibiting HUVEC proliferation are shown in Table 37.

[0330] Table 36 IC50 and maximum inhibition rate of fusion construct V1SA-3005Hz6-V1SA in HUVEC proliferation inhibition experiment

[0331] Table 37 IC50 and maximum inhibition rate of fusion construct 3005Hz6-2V1SA in HUVEC proliferation inhibition experiment

[0332] The results in Tables 36 and 37 show that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA were both able to effectively block the downstream signaling pathways activated by human VEGFA165 at the cellular level, inhibiting the proliferation of HUVECs stimulated by human VEGFA165, with activity similar to that of the positive control, aflibercept (a receptor drug). Furthermore, the results showed that whether or not the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA were pre-incubated with HSA had no significant effect on their HUVEC proliferation inhibitory activity.

[0333] Example 9 Study on the Inhibition of VEGFR2 Phosphorylation Induced by VEGFA by Fusion Constructs

[0334] This example uses primary HUVEC cells to verify the inhibitory effect of the fusion construct on VEGFA-induced VGFR2 phosphorylation.

[0335] In the experiment, primary HUVEC cells were digested and centrifuged, and then resuspended in ECM culture medium (containing 5% FBS, 1% epidermal growth factor, and 1% penicillin-streptomycin) (purchased from Zhongqiao Xinzhou, product number: ZQ-1304) at a concentration of 3×10 5 Cells were plated at a density of 100 μL / mL in a 96-well plate, with 100 μL of cell suspension per well. The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0336] VEGFA165 and fusion constructs were prepared in ECM medium. Untagged human VEGFA165 (purchased from GenScript, Cat. No. Z03073) at a working concentration of 50 ng / mL (1.3 nM) was premixed with varying concentrations of the fusion constructs V1SA-3005Hz6-V1SA (pre-incubated with HSA at the same molar concentration) and 3005Hz6-2V1SA (pre-incubated with HSA at the same molar concentration). VEGFA165-free and VEGFA165-only controls were also prepared. The mixture was incubated at 37°C for 30 minutes before being added to HUVEC cells plated overnight and incubated for 5 minutes. The cells were washed once with PBS, and 65 μL of strong RIPA lysis buffer (purchased from Beyotime, Cat. No. P0013B) containing PMSF was added to each well and lysed on ice for 20 minutes. Transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 13,000 rpm for 15 minutes (4°C). Collect the supernatant. Add 5× Loading buffer and heat at 95°C for 6 minutes. Collect the supernatant for western blot analysis to detect VEGFR2 phosphorylation. For immunoblotting, the anti-GAPDH antibody for detecting the internal control GAPDH was purchased from Proteintech, catalog number 60004-1-Ig. The antibody for detecting VGFR2 phosphorylation at tyrosine 1175 (p-VEGFR2) was purchased from Cell Signaling Technology, catalog number 2478S.

[0337] The signal intensities of the phosphorylated VEGFR2 (p-VEGFR2) band and the GAPDH band in the immunoblotting experiment were quantified, and the signal intensity ratio of the two bands (p-VEGFR2 / GADPH) was calculated to obtain the inhibition rate of the tested protein on VEGFR2 phosphorylation.

[0338] The calculation formula for the inhibition rate of VEGFR2 phosphorylation by the test protein is:

[0339] The inhibition rate of the fusion construct V1SA-3005Hz6-V1SA on VEGFR2 phosphorylation is shown in Table 38.

[0340] Table 38 Inhibition rate of VEGFR2 phosphorylation by fusion construct V1SA-3005Hz6-V1SA

[0341] Table 38 shows that V1SA-3005Hz6-V1SA can effectively inhibit human VEGFA-induced VEGFR2 phosphorylation in HUVEC cells. When the molar concentration of V1SA-3005Hz6-V1SA (incubated with HSA) is 5 times that of VEGFA, the inhibition rate of VEGFR2 phosphorylation can reach more than 90%.

[0342] The inhibition rate of the fusion construct 3005Hz6-2V1SA on VEGFR2 phosphorylation is shown in Table 39.

[0343] Table 39 Inhibition rate of fusion construct 3005Hz6-2V1SA on VEGFR2 phosphorylation

[0344] Table 39 shows that 3005Hz6-2V1SA can effectively inhibit human VEGFA-induced VEGFR2 phosphorylation in HUVEC cells. When the molar concentration of 3005Hz6-2V1SA (incubated with HSA) is 3 times that of VEGFA, the inhibition rate of VEGFR2 phosphorylation can reach more than 90%.

[0345] Example 10 Study on the activity of fusion constructs in VEGF reporter gene cells

[0346] This example uses a VEGF reporter gene system to evaluate the activity of the fusion construct in blocking the VEGF downstream signaling pathway.

[0347] HEK-293 / VEGF / NFAT stable reporter gene cell line (purchased from China Food and Drug Inspection Institute) is transfected with HEK-293 cells to co-express VEGFR2 (KDR) and NFAT-RE-luc 2p genes. When VEGFA recognizes and activates the KDR receptor, it initiates the intracellular downstream signaling pathway, activates the transcription factor NFAT, and initiates the expression of the luciferase reporter gene. At this time, chemiluminescence can be generated after the addition of luciferase substrate.

[0348] HEK-293 / VEGF / NFAT reporter gene cells were digested and centrifuged, and then resuspended in DMEM containing 1% FBS at a cell density of 5 × 10 4ng / mL, plated in a white-bottomed 96-well plate (purchased from Costar, Catalog No. 3917), 80 μL per well, and incubated overnight at 37°C in a 5% CO2 incubator. The next day, VEGFA165 (purchased from GenScript, Catalog No. Z03073) was prepared in DMEM containing 1% FBS to 80 ng / mL and mixed with varying concentrations of V1SA-3005Hz6-V1SA (with or without preincubation with an equimolar concentration of HSA) or 3005Hz6-2V1SA (with or without preincubation with an equimolar concentration of HSA) and incubated at 37°C for 30 minutes. A negative control consisted of no VEGFA165, and a positive control consisted of VEGFA165 alone. 20 μL of the mixture (test protein and VEGFA165) was added to 80 μL of cells and incubated at 37°C for 6 hours. 100 μL of luciferase reporter gene substrate (purchased from Yeasen, catalog number: 11404ES60) was added to each well, and the relative light unit (RLU) of luciferin was measured using a multifunctional microplate reader (purchased from Agilent BioTek, model: Synergy H1).

[0349] Under the above experimental conditions, the activity detection results of the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA in inhibiting the luminescence of the fluorescein substrate are shown in Figure 17(A) and Figure 17(B), respectively, and the fitted IC50 are shown in Table 40 and Table 41, respectively.

[0350] Table 40 Summary of IC50 inhibition of luciferin luminescence by the fusion construct V1SA-3005Hz6-V1SA in the VEGF reporter gene system with or without pre-incubation of HSA

[0351] Table 41 Summary of IC50 inhibition of luciferin luminescence by the fusion construct 3005Hz6-2V1SA in the VEGF reporter gene system with or without pre-incubation of HSA

[0352] The results in Figure 17 and Tables 40 and 41 show that both the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can block activation of downstream signaling pathways stimulated by VEGFA165 at the cellular level, thereby reducing nuclear import of the transcriptional promoter NFAT and activation of the Luciferase reporter gene. Their IC50 values ​​were both 0.24 nM. In this example, whether or not the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA were pre-bound to HSA had little effect on their blocking activity on VEGFA downstream signaling.

[0353] Based on the above-mentioned activity detection experiments at the protein and cell levels, the fusion constructs V1SA-3005Hz6-V1SA (SEQ ID NO: 32) and 3005Hz6-2V1SA (SEQ ID NO: 33) composed of the bivalent anti-VEGFA nanobody (V1-SA1, SEQ ID NO: 20) and the humanized anti-albumin nanobody (3005Hz6, SEQ ID NO: 9) both have strong HSA binding activity and affinity for VEGFA. The fusion constructs can effectively inhibit VEGFA165-induced HUVEC proliferation and VEGFR2 phosphorylation at the cellular level, and inhibit the fluorescein luminescence of VEGF reporter gene cells. These data indicate that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA have strong VEGFA neutralizing activity, can effectively block the VEGFA downstream signaling pathway, inhibit VEGFA-induced cell proliferation, and can potentially be used to treat diseases related to VEGFA-induced abnormal vascular proliferation, such as wet AMD, diabetic macular edema, tumors, etc.

[0354] The data also demonstrate that the antibody components within the fusion constructs, which bind to different antigens, can function independently, with minimal crosstalk between the components. Binding to HSA does not affect the biological function of the effector molecules. Therefore, it is expected that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA will function normally in blocking VEGFA signaling and inhibiting endothelial cell proliferation upon albumin binding in vivo.

[0355] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0356] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An anti-VEGFA fusion construct, characterized in that The fusion construct comprises an anti-albumin antibody or an antigen-binding fragment thereof and an anti-VEGFA antibody or an antigen-binding fragment thereof, wherein the anti-VEGFA antibody or an antigen-binding fragment thereof is linked to the anti-albumin antibody or an antigen-binding fragment thereof.

2. The fusion construct according to claim 1, characterized in that The anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein, The amino acid sequence of CDR-H1 comprises SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1; the amino acid sequence of CDR-H2 comprises SEQ ID NO:41 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:41; The amino acid sequence of CDR-H3 comprises SEQ ID NO: 3 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

3.

3. The fusion construct according to claim 1 or 2, characterized in that X1X2 in SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG) represents DS, DA, EG or DG.

4. The fusion construct according to any one of claims 1 to 3, characterized in that The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 include any of the following groups: A) SEQ ID NO: 1, 2, 3; B) SEQ ID NO: 1, 10, 3; C) SEQ ID NO: 1, 11, 3; D) SEQ ID NO: 1, 12, 3.

5. The fusion construct according to claim 1, wherein The anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein, the amino acid sequence of CDR-H1 comprises SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 16; the amino acid sequence of CDR-H2 comprises SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 17; The amino acid sequence of CDR-H3 comprises SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

18.

6. The fusion construct according to any one of claims 1 to 5, characterized in that The anti-albumin antibody or antigen-binding fragment thereof or anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence, wherein the modification site of the humanized sequence is located in a non-CDR region. Preferably, the modification site of the humanized sequence is located in the framework region and / or constant region of the antibody.

7. The fusion construct according to any one of claims 1 to 6, characterized in that The structures of the anti-albumin antibody or its antigen-binding fragment or anti-VEGFA antibody or its antigen-binding fragment include nanobodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, F(ab')2 fragments, single-chain antibodies or linear antibodies.

8. The fusion construct according to any one of claims 1 to 7, characterized in that The amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 4, 6-9, 13-15, or has at least 80% identity with any one of SEQ ID NOs: 4, 6-9, 13-15.

9. The fusion construct according to any one of claims 1 to 7, characterized in that The amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 19-28, or has at least 80% identity with any one of SEQ ID NOs: 19-28.

10. The fusion construct according to any one of claims 1 to 9, characterized in that The anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are directly or indirectly connected. Preferably, the indirect connection can be through a linker, a functional domain and / or a linker for coupling; wherein the linker is selected from a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin or avidin; The functional domain is a combination of one or more of an Fc fragment, serum albumin, cytokine, transferrin or a scaffold protein; the linker for coupling includes a functional group linker, preferably, the functional group linker includes a thiol, amino, hydroxyl and / or carboxyl reactive group, More preferably, the fusion construct may comprise one or more anti-VEGFA antibodies or antigen-binding fragments thereof, wherein the anti-VEGFA antibodies or antigen-binding fragments thereof are directly or indirectly linked to the N-terminus, C-terminus and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof, Further preferably, the fusion construct comprises any one of SEQ ID NOs: 29-35, or has at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 29-35.

11. A nucleic acid, characterized in that The nucleic acid encodes the fusion construct according to any one of claims 1 to 10.

12. The nucleic acid according to claim 11, characterized in that The nucleic acid comprises any one of the nucleotide sequences in SEQ ID NOs: 62-71 or a degenerate sequence thereof, or a nucleotide sequence that is at least 80% identical to any one of the nucleotide sequences in SEQ ID NOs: 62-71 and has the function of encoding an anti-VEGFA antibody or an antigen-binding fragment thereof.

13. The nucleic acid according to claim 11 or 12, characterized in that The nucleic acid further comprises any one of the nucleotide sequences in SEQ ID NOs: 45, 47-50, 56-58 or a degenerate sequence thereof, or a nucleotide sequence that is at least 80% identical to any one of the nucleotide sequences in SEQ ID NOs: 45, 47-50, 56-58 and has the function of encoding an anti-albumin antibody or an antigen-binding fragment thereof.

14. The nucleic acid according to any one of claims 11 to 13, characterized in that The nucleic acid comprises any one of nucleotide sequences in SEQ ID NOs: 72-78 or a degenerate sequence thereof, or has at least 80% identity with any one of nucleotide sequences in SEQ ID NOs: 72-78, and has a nucleotide sequence encoding the function of the fusion construct.

15. A carrier, characterized in that The vector comprises the nucleic acid according to any one of claims 11 to 14.

16. A host cell, characterized in that The host cell comprises the nucleic acid according to any one of claims 11 to 14 or the vector according to claim 15.

17. A method for preparing the fusion construct according to any one of claims 1 to 10, characterized in that: The preparation method comprises culturing the host cell according to claim 16 to express the fusion construct.

18. Use of the fusion construct according to any one of claims 1 to 10, the nucleic acid according to any one of claims 11 to 14, the vector according to claim 15 or the host cell according to claim 16 in the preparation of a product for treating and / or diagnosing a disease.

19. A product for treating and / or diagnosing a disease, characterized in that The product for treating and / or diagnosing a disease comprises any of the following: A) the fusion construct of any one of claims 1 to 10; B) the nucleic acid according to any one of claims 11 to 14; C) the vector according to claim 15; or D) The host cell according to claim 16.

20. A method for detecting VEGFA, characterized in that: The detection method comprises contacting a sample to be tested with the fusion construct according to any one of claims 1 to 10, and then detecting the content of the complex formed by VEGFA and the fusion construct. Preferably, the VEGFA is from a mammal, more preferably, the mammal is from a human, mouse or monkey.